WO2007000199A1

WO2007000199A1 - Method and device for optically scanning a sample

Info

Publication number: WO2007000199A1
Application number: PCT/EP2006/003227
Authority: WO
Inventors: Jürgen TÜMPNER
Original assignee: Olympus Soft Imaging Solutions Gmbh
Priority date: 2005-06-24
Filing date: 2006-04-08
Publication date: 2007-01-04
Also published as: DE102005029381A1; US20090091823A1; EP1893940A1; DE102005029381B4; JP2008547052A

Abstract

The invention relates to a method and a device for optically scanning a sample, especially using a microscope. To this end, an adjustment unit (2, 3) and a scanning device (4, 5) are provided. The sample (1) is displaced in relation to the scanning device (4, 5) by means of the adjustment unit (2, 3) which is acted upon by a control installation (7), or vice versa. According to the invention, a displacement window (F) is defined for the adjustment unit (2, 3) and/or the scanning device (4, 5), inside which mechanical collisions between the sample (1) and the scanning device are prevented. This is especially advantageous in biological samples. A non-contact sample sensor (8) is provided for the prevention of collisions, said sensor operating, for example, by electromagnetic or acoustic waves.

Description

Method and apparatus for optically scanning a sample

Description:

The present invention relates to a method for the optical scanning of a sample, comprising an adjusting unit and a scanning device, according to which the

Probe is moved by means of the acted upon by a control unit adjusting unit relative to the scanning unit, or vice versa, and then by means of a sample sensor, a movement window for the adjustment and / or the

Scanning is defined within which mechanical collisions between the sample and the scanner are excluded.

A method of the embodiment described above is presented in DE 102 39 794 A1. This is about a measuring device that is equipped with a protective device, which prevents a moving part colliding with an object. The protector includes a protector body and a sensor protruding from the protector body over a predetermined length to sense a distance between the object and the protector body by its elastic deformation upon contact with the object.

Comparable is the JP 022 47 967 A before. For here an electrode is realized, which closes an electrical circuit in contact with a sample and thus emits an alarm to prevent further movement of the adjustment.

In the context of AT 197 096 finally a microscope objective with protective device is presented. In this case, a part carrying the optic is displaceable relative to a socket part in the axial direction against spring force. During the displacement of the optics-carrying part, a contact is actuated, which in turn triggers an acoustic or an optical signal. In the prior art, it is fundamentally necessary for mechanical contact to be established between the different and differently designed sample sensors and the sample. This is disadvantageous in the case of highly sensitive specimens, because they can be damaged thereby, even if only slight contact is made.

Generally, for optical scanning of the sample, it is mostly examined for transmission. In principle, reflection measurements are also possible. The respective sample may be a biological sample, a cell section or even a material science sample, such as a material cut.

Usually, the scanning device always captures only a specific section of the sample with the desired resolution and consequently generates a single image. The adjustment unit now ensures that the respective individual images taken with the scanning device are combined to form at least one overall image in the control system. This is not mandatory.

In any case, the scanner has mostly an optical unit and a recording unit. The optical unit is not limited to one or more microscope objectives, while the recording unit is embodied, for example, as a CCD chip (CCD = Charge Coupled Device) or includes such. The recording unit or the CCD chip is regularly in the image plane of the associated microscope objective to record the individual image and transfer it to the control system. The control system in turn reads the recording unit and stores the respective frame. After all the individual images have been acquired, they are combined to form the one or more overall images. Usually, the sample is moved with the aid of the adjusting unit relative to the scanning device. For this purpose, the sample is regularly held by a sample table, which in turn is moved via adjusting or adjusting devices. It usually takes place not only a movement in X- / Y-level, to record the respective frames and store in the control system. In fact, the adjustment unit usually also provides for an adjustment in the Z direction, that is to say the height direction, which is always required when, for example, focusing is to be effected with the aid of the microscope objective or the optical unit. The focusing can be done both automatically and manually. In the former case, the image contrast is examined with the aid of gray value deviations and the picture with the highest contrast is classified as belonging to the focus. In addition, of course, a manual focus is possible. Likewise, the invention comprises modifications in such a way that it is not the adjusting unit or the sample table that is moved relative to the scanning device, but the scanning device additionally or alternatively with respect to the adjusting unit. Of course, both are possible, as the latter application makes clear.

Then, the sample table is moved in the X / Y plane, whereas the scanning device undergoes a movement in the Z direction. In such a variant and otherwise there is a risk that the sample is accidentally damaged. This is particularly painful in the case that the sample is a particularly valuable individual piece or a living cell culture, which can be irreparably destroyed by such an operation. This is where the invention starts.

The invention is based on the technical problem of further developing a method of the embodiment described at the beginning in such a way that sample preparation Damage can be excluded in any case. In addition, a correspondingly suitable device should be specified.

To solve this technical problem, the invention proposes in a generic method for the optical scanning of a sample, that the sample sensor operates without contact, preferably with reference to electromagnetic waves and / or sound waves. In the former case, both distance measurements (interferometric) and time measurements (transit time of a pulse) are conceivable. Similarly, work is done on sound waves.

Accordingly, a movement window is usually specified for the adjustment unit and / or the scanning device, usually within the control system, so that the adjustment unit and / or the scanning device can only be moved within this movement window or an alarm message is issued when the movement window is exited. The movement window is determined and specified with the aid of the non-contact sample sensor.

It has proven useful if the motion window is set depending on various parameters. These parameters may not be limited to the size of the sample, the initial position and speed of the adjustment and / or scanning device (4, 5) and possibly their design. Depending on this, the movement window is set variably in each case.

The sample sensor defines - as already mentioned - said movement window. In this case, the sample sensor preferably reports, once or continuously, distance measurement values between the sample and / or the adjustment unit and the scanning device to the control system. In fact, the sample sensor may, for example, sample the size of the sample and transfer associated sample dimension values to the control system. If, in addition, the initial position of the adjusting unit and, for example, the design of the sample receiving sample stage are stored in the control system, a free space between the sample and the scanning device can be easily deduced in connection with said sample dimension values. This free space can be equated with the maximum possible motion window. As a rule, the movement window limits movements of the adjustment unit and / or the scanning device in one dimension, usually the Z direction. If the free space between the sample and the scanner is known, this can be converted directly into values for the movement window in this Z direction. For safety reasons, the motion window will usually be much smaller than the previously mentioned free space. Values of 50% to 80% are conceivable here. That is, the motion window occupies 50% to 80% of the free space.

In addition to the sample sensor, it has proven useful if a displacement sensor is additionally implemented. With the help of this adjustment sensor movements of the adjustment unit can be detected - starting with their previously determined initial position. In the example case, the adjustment sensor serves to record and evaluate movements of the adjustment unit in the Z direction. If the movement window is fixed in the Z direction, it can be determined directly with the aid of the adjustment sensor whether the adjustment unit or the sample moves in the direction of one of the two boundaries of the movement window or not. In this case, the movement window is in each case variably adapted to the movement of the sample relative to the scanning device. As a result, of course, the distance measurement values between the sample and the scanner and, of course, the size of the free space change as well. If the sample moves toward a boundary of the movement window, an alarm signal, for example, is emitted acoustically and / or optically. However, it is also possible to proceed in such a way that, when a limit of the movement window is reached, the sample can no longer be moved further with the aid of the adjustment unit, thus blocking the adjustment unit.

Since the sample sensor operates without contact, it can detect the free space between the sample and the scanning device in a corresponding manner, without the sample being damaged. The sample sensor may work with (ultra-) sound waves and / or electromagnetic waves. Conceivable here are continuous processes, for example with the aid of an interferometer, to measure the free space and derive the motion window therefrom. However, it is also possible to work with wave pulses which, for example, are emitted by the scanning device and reflected by the sample. From their runtime or an associated time measurement, the free space can be determined and consequently derive the motion window. In addition, the sample sensor is usually capable of scanning the sample for its sample dimensions.

The adjustment sensor is usually associated with one or more adjustment of the adjustment. In the event that the adjustment sensor is intended to detect a movement of the scanning device or of the microscope objective in the Z direction, the adjustment sensor is usually assigned to a handwheel or a corresponding Z drive provided at this point. In the former case, the adjustment can be designed as a rotary encoder. In the latter case, it may be a displacement sensor or the like.

As a result, a method is provided which, by defining a motion window for the adjustment unit, ensures that mechanical Collisions between the sample and the scanner can be reliably excluded. This is achieved essentially by the fact that the free space between the sample and the scanning device is determined without contact and serves as the basis for defining a variable movement window.

The non-contact sample sensor based on preferably electromagnetic waves and / or (ultra-) sound waves ensures that the associated sample is not damaged. Because in contrast to the prior art, for example according to DE 102 39 749 A1, a mechanical contact is omitted in any case. This circumstance is of particular importance against the background that even the tiniest touch can irreparably damage most biological samples. This is excluded in the context of the invention in any case.

In addition, the sample sensor according to the invention can not simply work without contact, but it represents an advantageous embodiment, as it were a lateral image of the sample or its shadow. Here, the invention is based on the further knowledge that the accuracy of the determination of the Free space and therefore of the movement window.

In fact, the distance between the sample and the scanner during adjustment changes permanently and can be put on an even more reliable basis by this procedure. For the determination of the shadow cast can be determined with a stationary sample sensor which detects the free space between the sample and the scanning device from the side. In contrast, the sample sensor is otherwise placed either in the scanning device and / or the adjustment, so that changes in their distance from each other by the in Direction of the change in distance and not perpendicular to this (as in the shadow) working sample sensor can be determined.

The invention also provides a device for optically scanning a sample as described in claim 6. Advantageous embodiments of this device are treated in claims 7 to 10.

In the following the invention will be explained in more detail with reference to a drawing showing only one exemplary embodiment; show it:

Fig. 1 shows a device according to the invention schematically and

2 and 3 are each an enlarged detail of FIG. 1.

In the figures, a device for optical scanning of a sample 1 is shown, which is not limited to a transparent biological tissue section. This is illuminated by means of a white light source W or the like, which is located below an adjustment unit 2, 3. The adjusting unit 2, 3 is composed of adjusting devices 2 and a sample table 3.

With the help of the adjusting devices 2, the sample table 3 can be moved in the example case in the X / Y direction, so that different areas of the sample 1 can be recorded. In addition, the sample table 3 can optionally also be adjusted in the Z direction. This is not shown. For a relative movement between the sample 1 and a scanning device 4, 5 in the Z direction is accomplished in the present case, that the scanning device 4, 5 is associated with a further adjusting device 6, in which it is not limiting to a handwheel 6 in conjunction with a Z Actuator acts. The scanning device 4, 5 is composed of a plurality of objectives or microscope objectives 4 as an optical unit 4, which can image different sized image sections of the sample 1 to a recording unit 5 selectively. The recording unit 5 is a CCD chip with, for example, 1 million pixels. The image of the transmitted sample 1 generated on the CCD chip is recorded in a control system 7 as a single image E. As indicated, the control unit 7 combines a plurality of individual images E into an overall image.

The control system 7 also controls the adjusting device 2 or the adjusting unit 2, 3 and optionally the optical unit 4 by a desired lens 4 is selected. In addition, the control system 7 also controls the adjusting device 6 in the Z direction. In fact, the illustrated device with the aid of the adjusting device 6 enables both automatic focusing of the sample 1 with the aid of the selected objective 4 and manual focusing with the illustrated handwheel 6.

When focusing a distance A between the sample 1 and the scanning device 4, 5 is changed by means of the adjusting device 6. Starting from the illustrated starting position of the adjusting unit 2, 3 with respect to the scanning device 4, 5, this distance A is a maximum possible movement window F for the adjusting unit 2, 3, within which mechanical collisions with the scanning device 4, 5 are excluded (see FIG 3). In fact, the movement window F is usually set smaller than the said (maximum) distance A, for example, the movement window F is 80% of the distance A, so that the following applies:

(1) F = 0.8A. Of course, other values are also conceivable, so that the following range is advantageously covered:

(2) 0.5A ≦ F <1.0A

In order to variably determine and record the movement window F or the distance A in detail, the illustrated device has at least one sample sensor 8 which records once or continuously distance measurement values, ie the distance A, between the sample 1 and / or the adjustment unit 2. 3 and the scanning device 4, 5 reports to the control system 7. In addition, the sample 1 can be scanned with the aid of the sample sensor 8 with regard to its sample dimensions. This is usually the case.

In the example of FIG. 1, the sample sensor 8 is arranged on a stationary holding arm for the scanning device 4, 5 or in this. If the selected lens 4 is known and also its dimensions, it can be determined from a projection Ü of the objective 4 with respect to a lower edge of the holding arm according to the distance of the lower edge of the lens 4 from

Sample sensor 8 to the distance of the lens 4 relative to the sample table

3 infer as follows:

(3) A = A - O - H,

where H indicates the "height" of the sample 1 above the sample table 3 and has been previously determined, for example. Of course, the sample sensor 8 may also be attached directly (head-side) to the objective 4 and continuously transmit the distance A to the control system 7. As a further alternative, the sample sensor 8 scans the sample 1 with respect to its sample dimensions, which also happens without contact. For this purpose, the sample sensor 8 can laterally illuminate the sample 1 and close it from the shadow image at its height "H" (see FIG. 3). However, in the illustrated embodiment, the sample sensor 8 is fixedly attached to the scanner 4, 5, or lens 4, and directly measures the distance A from its mounting location to a head of the sample 1. This head usually coincides with the surface of a cover glass. wherein the sample 1 is placed between said coverslip and a slide, as Fig. 3 makes clear.

In any case, the sample sensor 8 is able to determine the height H of the sample 1 in comparison to the surface of the sample table 3 and also the distance A of the sample 1 to the lower edge of the lens 4 and possibly the distance A ¹ . Assuming that in the control system 7 is known which lens 4 is currently used, and taking into account the associated dimensions, in particular the supernatant Ü, with the help of the height H can be directly deduced the distance A, if, for example the sample sensor 8 measures the distance A ¹ . For this distance A results simply from the distance of the selected objective 4 or its projection U and its distance from the surface of the sample table 3 minus the height H of the sample 1 in accordance with equation (3) given above.

Based on this determined variable distance A, the control system 7 now predefines the movement window F according to the previously specified rule (1) or (2). In order to take dynamic movements of the scanning device 4, 5 with respect to the sample 1, an adjustment sensor 9 is additionally realized in addition to the sample sensor 8, which detects movements of the adjustment unit 2, 3. In the exemplary embodiment, the adjustment sensor 9 is the handwheel 6 or the Adjusting device 6 assigned to the movement of the scanning device 4, 5 in the Z direction.

In fact, the adjustment sensor 9 is a rotation angle sensor which detects movements of the handwheel 6 or the additionally or alternatively provided adjustment drive 6 '. Depending on how the scanning device 4, 5 - starting in its initial position - moves relative to the sample table 3 and consequently the sample 1, calculates the control system 7 from the associated adjustment - recorded using the Verstellsensors 9 - the remaining distance A between the microscope objective 4 and the sample 1 and its head. For this purpose, the distance A needs only once in the initial position of the scanning device 4, 5 are determined relative to the sample 1. All changes in the distance A are then taken into account via the adjustment sensor 9 and the control system 7 via the adjustment. In order to increase safety, however, as a rule the distance A is continuously measured and tested for conformity with the values calculated in the control system 7 via the adjustment path and the displacement sensor 9.

If there is a deviation, an alarm signal is output and / or the adjusting device 6 is blocked by means of the control system 7. The same occurs if the adjustment unit 2, 3 moves to the edge of the movement window F or reaches it. Depending on the speed of adjustment in the Z direction, it is also possible to work with multipliers other than 0.8 in equations (1) and (2) in order to derive the motion window F from the distance A. It is conceivable to reduce the factor to 0.5 at high speeds of the scanning device 4, 5 in the Z direction. On the other hand, a slow adjustment in the Z direction may also give values of up to almost 1.0. Achieved in this process so the scanning device 4, 5 a minimum distance A relative to the sample 1 and a lower limit of the movement window F, the handwheel 6 is empty or it is issued a warning signal. In fact, the hand wheel 6 is not mechanically coupled directly to an actuator or the like, but rather rotational movements of the handwheel 6 with the adjustment sensor 9 are detected and tapped and then converted into adjusting movements of the additionally provided actuating drive 6 'with the aid of the control system 7. Previously, these adjusting movements of the handwheel 6 and thus adjustment of the Verstellsensors 9 have been evaluated in the control system 7, to the extent that the adjoining adjustment path is possible or not. If the minimum distance A between the sample 1 and the microscope objective 4 is undershot (for example, 1 mm or another value, depending on the application), the control system 7 ensures in the example case that the actuator 6 'is only adjusted so far that the minimum distance is complied with.

In addition, and independent of the previously described mode of operation, the sample sensor 8 is not only able to determine the variable distance A with the aid of the control unit 7 and to specify the movement window F. Rather, the respective position of the scanning device 4, 5 with respect to the sample 1 in the control system 7 can be logged with the help of the sample sensor 8 in addition. If these logged values are simultaneously compared with those of the adjustment sensor 9, statements about the positioning accuracy of the handwheel 6 or of the additionally or alternatively provided positioning drive 6 'can be made.

It is also possible, if necessary, to make a correction from any deviations between the actually measured position of the scanning device 4, 5 in comparison to the specification according to the adjustment sensor 9. D. h., With the actuator 6 'and the handwheel 6 is a certain distance A of Scanning device 4, 5 set against the sample 1 and compared with the actually achieved and measured by the sample sensor 8 value. If this procedure is now carried out for different distances A, correction values K can be given in each case as a function of the distance A, corresponding to K (A).

Thus, the invention is able to dispense with particularly elaborate actuators 6 'or handwheels 6, because ultimately the sample sensor 8 in conjunction with the Verstellsensor 9 and the control system 7 for a corresponding correction and compensation possibly (mechanical) inaccurate - worries.

Claims

claims:

1. A method for optical scanning of a sample (1), comprising an adjusting unit (2, 3) and a scanning device (4, 5), after which the sample (1) by means of the control unit (7) acted upon adjusting unit (2, 3) relative to the scanning device (4, 5) is moved, or vice versa, and then by means of a sample sensor (8) a movement window (F) for the adjusting unit (2, 3) and / or the scanning device (4, 5) is defined within which mechanical collisions between the sample (1) and the scanning device (4, 5) are excluded, characterized in that the sample sensor (8) without contact, for example, with reference to electromagnetic and / or sound waves works.

2. The method according to claim 1, characterized in that the movement window (F) depending on various parameters such as the size of the sample

(1), the initial position and speed of the adjusting unit (2, 3) and / or the scanning device (4, 5) and optionally their configuration is set variably.

3. The method according to claim 1 or 2, characterized in that the movement window (F) limits movements of the adjusting unit (2, 3) and / or the scanning device (4, 5) in one dimension.

4. The method according to any one of claims 1 to 3, characterized in that the sample sensor (8) once or continuously distance measurement values between the sample (1) and / or the adjusting unit (2, 3) and the scanning device (4, 5) to the Control system (7) reports.

5. The method according to any one of claims 1 to 4, characterized in that in addition to the sample sensor (8) an adjustment sensor (9) is realized, which detects movements of the adjustment unit (2, 3).

6. An apparatus for optical scanning of a sample (1), comprising an adjusting unit (2, 3) and a scanning device (4, 5), wherein the sample (1) is acted upon by the adjusting device (2, 3) acted upon by a control system (7). relative to the scanning device (4, 5) is moved, or vice versa, and then by means of a sample sensor (8) a movement window (F) for the adjusting unit (2, 3) and / or the scanning device (4, 5) is defined within which mechanical collisions between the sample (1) and the scanning device (4, 5) are excluded, characterized in that the sample sensor (8) operates without contact, using, for example, electromagnetic and / or sound waves.

7. The device according to claim 6, characterized in that the sample sensor (8) detects between the sample (1) and the scanning device (4, 5) existing free space.

8. Apparatus according to claim 6 or 7, characterized in that the sample sensor (8) additionally or alternatively scans the sample (1).

9. Device according to one of claims 6 to 8, characterized in that in addition to the sample sensor (8), an adjustment sensor (9) is provided.

10. Device according to one of claims 6 to 9, characterized in that the adjustment sensor (9) one or more adjusting devices (2, 6) of the adjusting unit (2, 3) is assigned.